Recombinant Saccharomyces cerevisiae expressing P450 in artificial digestive systems: a model for biodetoxication in the human digestive environment

The use of genetically engineered microorganisms such as bacteria or yeasts as live vehicles to carry out bioconversion directly in the digestive environment is an important challenge for the development of innovative biodrugs. A system that mimics the human gastrointestinal tract was combined with a computer simulation to evaluate the survival rate and cinnamate 4-hydroxylase activity of a recombinant model of Saccharomyces cerevisiae expressing the plant P450 73A1. The yeasts showed a high level of resistance to gastric and small intestinal secretions (survival rate after 4 h of digestion, 95.6% +/- 10.1% [n = 4]) but were more sensitive to the colonic conditions (survival rate after 4 h of incubation, 35.9% +/- 2.7% [n = 3]). For the first time, the ability of recombinant S. cerevisiae to carry out a bioconversion reaction has been demonstrated throughout the gastrointestinal tract. In the gastric-small intestinal system, 41.0% +/- 5.8% (n = 3) of the ingested trans-cinnamic acid was converted into p-coumaric acid after 4 h of digestion, as well as 8.9% +/- 1.6% (n = 3) in the stomach, 13.8% +/- 3.3% (n = 3) in the duodenum, 11.8% +/- 3.4% (n = 3) in the jejunum, and 6.5% +/- 1.0% (n = 3) in the ileum. In the large intestinal system, cinnamate 4-hydroxylase activity was detected but was too weak to be quantified. These results suggest that S. cerevisiae may afford a useful host for the development of biodrugs and may provide an innovative system for the prevention or treatment of diseases that escape classical drug action. In particular, yeasts may provide a suitable vector for biodetoxication in the digestive environment.     

Translokin is an intracellular mediator of FGF-2 trafficking

Basic fibroblast growth factor (bFGF or FGF-2) exerts its pleiotropic activities both as an exogenous and an intracellular factor. FGF-1 and FGF-2 are prototypes for this dual signalling, but the mechanisms of their intracellular actions remain unknown. Here we show that Translokin, a cytoplasmic protein of relative molecular mass 55,000 (M(r) 55K), interacts specifically with the 18K form of FGF-2. Translokin is ubiquitously expressed and colocalizes with the microtubular network. As Translokin does not interact with FGF-1, we used a strategy based on FGF-1-FGF-2 chimaeras to map the interacting regions in FGF-2 and to generate Nb1a2, a non-interacting variant of FGF-2. Although most of the FGF-2 properties are preserved in Nb1a2, this variant is defective in intracellular translocation and in stimulating proliferation. The fusion of a nuclear localization signal to Nb1a2 restores its mitogenic activity and its nuclear association. Inhibiting Translokin expression by RNA interference reduces the translocation of FGF-2 without affecting the intracellular trafficking of FGF-1. Our data show that the nuclear association of internalized FGF-2 is essential for its mitogenic activity and that Translokin is important in this translocation pathway.     

Oxidative damage generated by an oxo-metalloporphyrin onto the human telomeric sequence

The cationic metalloporphyrin Mn-TMPyP activated by KHSO(5) has been used as cleaver of an oligonucleotide containing the four human telomere repeats of 5'-GGGTTA. This oligonucleotide formed an intramolecular quadruplex DNA under 200 mM KCl as probed by DMS footprinting and could fold into different quadruplex structures under 200 mM NaCl. We found that the oxo-metalloporphyrin was able to mediate efficient oxidative cleavage of the quadruplex. The location of damage showed that the metalloporphyrin was able to bind to the last G-tetrad of the quadruplex structure via an external interaction. This metalloporphyrin-G-tetrad interaction needs a relatively high flexibility of the single-stranded linker regions to allow the partial stacking of the metalloporphyrin with the last G-tetrad planar structure. The oxidative damage consisted of guanine oxidation within the interacting G-tetrad together with an 1'-carbon hydroxylation of deoxyribose residues of the thymidine residues located on the neighboring single-stranded loop. So the high-valent oxo-metalloporphyrin is able to mediate both electron-abstraction or H-abstraction on G or T residues, respectively, within the DNA quadruplex target.     

Protonmotive Mechanism of Heme-Copper Oxidases

The mechanism of coupling of proton and electron transfer in oxidases is reviewed and related to the structural information that is now available. A glutamate trap mechanism for proton/electron coupling is described.     

pH as a proxy for estimating plant-available Si? A case study in rice fields in Karnataka (South India)

Background and aims

Soil nutrient dynamics are affected by root-microbe interactions and plant development. We investigated the influence of plant growth stage and arbuscular mycorrhiza fungi (AMF) on carbon (C) and nitrogen (N) rhizodeposition and the transfer into the microbial biomass (MB).

Methods

Pea varieties (Pisum sativum L.) with (Frisson) and without mycorrhiza (P2) were ¹³C-¹⁵N-labelled and harvested at 45, 63, 71, and 95 days after sowing. Mycorrhization, MB, total C, N, ¹³C, ¹⁵N were determined in plant and soil compartments to calculate C and N derived from rhizodeposition (CdfR, NdfR).

Results

Total CdfR increased until pea maturity, NdfR until end of flowering. Their relative contribution steadily decreased over time, accounting for 4–10% of total plant C and N at harvest. Rhizodeposition contributed between 1 and 6% to MB C and N, although 20% of the rhizodeposits were discovered in the MB. Frisson released more NdfR than P2 but it was not possible to accurately estimate AMF effects on C and N due to differences in biomass partitioning.

Conclusions

CdfR followed an even flow from early growth until senescence. NdfR flow ceased after flowering possibly due to N relocation within the plant. Rhizodeposits contribute very little to MB in our study.

     

DNA cleavage studies of mononuclear and dinuclear copper(II) complexes with benzothiazolesulfonamide ligands

Copper-based transition metal complexes performing single- and double-strand scission of DNA have been studied. The dinuclear complexes [Cu(2)(L)(2)(OCH(3))(2)(NH(3))(2)] and [Cu(2)(L)(2)(OCH(3))(2)(DMSO)(2)] are more active than the corresponding mononuclear [Cu(L)(2)(py)(2)] (where HL= N-(4-methylbenzothiazol-2-yl)benzenesulfonamide), suggesting that the dinuclearity is an important factor in the oxidative cleavage of DNA. The cleavage efficiency of the complexes depends on the reducing agent used in the process, the tandem ascorbate/H(2)O(2) being the most efficient. PAGE analyses have shown that these complexes cleave DNA without sequence selectivity. The DNA degradation process takes place mainly by C1' oxidation, but C4' and C5' oxidations cannot be ruled out as minor pathways. These copper complexes preferably oxidize guanine under mild conditions, but under more drastic conditions the oxidation reactivity appears to be T>G>C>A, suggesting the intervention of hydroxyl radicals as active species.     

Comparative Analysis of Extracellular and Intracellular Proteomes of Listeria monocytogenes Strains Reveals a Correlation between Protein Expression and Serovar

Listeria monocytogenes, the etiologic agent of listeriosis, remains a serious public health concern, with its frequent occurrence in food environments coupled with a high mortality rate. Among the 13 serovars, human listeriosis is mostly associated with the serovar 4b, 1/2b, and 1/2a strains. To investigate the diversity of L. monocytogenes, the intracellular and extracellular proteins of 12 strains were analyzed by two-dimensional gel electrophoresis. These strains had different origins, belonged to different serovars (4b, 1/2a, and 1/2b), and presented with different levels of virulence in chicken embryos. The clustering of the strains in two groups based on proteomic patterns is in agreement with the L. monocytogenes phylogenetic lineages. Statistical analysis did not allow for identification of proteins specific to the isolate origin or the virulence level of the strains, but 26 and 21 protein spots were shown to be significantly overexpressed and underexpressed, respectively, in the six strains of serovar 1/2a (lineage II) compared to strains of serovar 1/2b or 4b. Moreover, a penicillin-binding protein was specific for serovar 1/2b and two protein spots identified as a serine protease were specific to serovar 4b. These protein spots, identified through peptide mass fingerprinting using matrix-assisted laser desorption ionization-time of flight mass spectrometry, were essentially found in the extracellular proteome and may have uses as potential markers for serotyping and risk analysis.     

Directed evolution predicts cytochrome b G37V target site modification as probable adaptive mechanism towards the QiI fungicide fenpicoxamid in Zymoseptoria tritici

Acquired resistance is a threat to antifungal efficacy in medicine and agriculture. The diversity of possible resistance mechanisms and highly adaptive traits of pathogens make it difficult to predict evolutionary outcomes of treatments. We used directed evolution as an approach to assess the resistance risk to the new fungicide fenpicoxamid in the wheat pathogenic fungus Zymoseptoria tritici. Fenpicoxamid inhibits complex III of the respiratory chain at the ubiquinone reduction site (Qi site) of the mitochondrially encoded cytochrome b, a different site than the widely used strobilurins which inhibit the same complex at the ubiquinol oxidation site (Q_o site). We identified the G37V change within the cytochrome b Q_i site as the most likely resistance mechanism to be selected in Z. tritici. This change triggered high fenpicoxamid resistance and halved the enzymatic activity of cytochrome b, despite no significant penalty for in vitro growth. We identified negative cross-resistance between isolates harbouring G37V or G143A, a Q_o site change previously selected by strobilurins. Double mutants were less resistant to both QiIs and quinone outside inhibitors compared to single mutants. This work is a proof of concept that experimental evolution can be used to predict adaptation to fungicides and provides new perspectives for the management of QiIs.     

Bilateral fractures of femoral neck in patients with moderate renal failure receiving fluoride for spinal osteoporosis.

Two patients with moderate renal failure sustained spontaneous bilateral hip fractures during treatment with fluoride, calcium, and vitamin D for osteoporosis. They had been taking sodium fluoride (40-60 mg/day) for 11 and 21 months, respectively. Histological examination of a specimen of the bone showed severe fluorosis in the first case, and quantitative analysis of bone showed osteomalacia and skeletal fluorosis in the other case. These abnormalities were considered to be the consequence of excessive retention of fluoride due to renal insufficiency. As bilateral femoral neck fractures are very rare these data suggest a causal link between fractures and fluoride in patients with renal failure. Thus fluoride should be given at a lower dosage, if at all, to patients with even mild renal failure.     

Recreation of neuronal Kv1 channel oligomers by expression in mammalian cells using Semliki Forest virus

The multiple roles of voltage-sensitive K(+) channels (Kv1 subfamily) in brain are served by subtypes containing pore-forming alpha (1.1-1.6) and auxiliary beta subunits, usually in an (alpha)(4)(beta)(4) stoichiometry. To facilitate structure/activity analysis, combinations that are prevalent in neurones and susceptible to alpha-dendrotoxin (alphaDTX) were reproduced in mammalian cells, using Semliki Forest virus. Infected Chinese hamster ovary cells expressed N-glycosylated Kv1.1 and 1.2 alpha subunits (M(r) approximately 60 and 62 K) that assembled and bound [(125)I]-alphaDTX with high affinity; an appreciable proportion appeared on the cell surface, with Kv1.2 showing a 5-fold enrichment in a plasma membrane fraction. To obtain 'native-like' alpha/beta complexes, beta1.1 or 2.1 (M(r) approximately 42 and 39 K, respectively) was co-expressed with Kv1.1 or 1.2. This slightly enhanced N-glycosylation and toxin binding, most notable with beta2. 1 and Kv1.2. Solubilization of membranes from cells infected with Kv. 1.2 and beta2.1, followed by Ni(2+) chromatography, gave a purified alpha1.2/beta2.1 complex with a size of approximately 405 K and S(20, W) = 15.8 S. Importantly, these values indicate that four alpha and beta subunits co-assembled as in neurones, a conclusion supported by the size ( approximately 260 K) of the homo-tetramer formed by Kv1.2 alone. Thus, an authentic K(+) channel octomer has been reconstructed; oligomeric species were also found in plasma membranes. To create 'authentic-like' hetero-oligomeric channels, Kv1.1 and 1.2 were co-expressed and shown to have assembled by the precipitation of both with IgGs specific for either. Consistently, confocal microscopy of cells labeled with these antibodies showed that the relatively low surface content of Kv1.1 was increased by Kv1.2. [(125)I]-alphaDTX binding to these complexes was antagonized by DTX(k), a probe selective for Kv1.1, in a manner that mimicks the pattern observed for the Kv1.1/1.2-containing channels in neuronal membranes.